One-carbon metabolism nutrients impact the interplay between DNA methylation and gene expression in liver, enhancing protein synthesis in Atlantic salmon.

Supplementation of one-carbon (1C) metabolism micronutrients, which include B-vitamins and methionine, is essential for the healthy growth and development of Atlantic salmon (Salmo salar). However, the recent shift towards non-fish meal diets in salmon aquaculture has led to the need for reassessments of recommended micronutrient levels. Despite the importance of 1C metabolism in growth performance and various cellular regulations, the molecular mechanisms affected by these dietary alterations are less understood. To investigate the molecular effect of 1C nutrients, we analysed gene expression and DNA methylation using two types of omics data: RNA sequencing (RNA-seq) and reduced-representation bisulphite sequencing (RRBS). We collected liver samples at the end of a feeding trial that lasted 220 days through the smoltification stage, where fish were fed three different levels of four key 1C nutrients: methionine, vitamin B6, B9, and B12. Our results indicate that the dosage of 1C nutrients significantly impacts genetic and epigenetic regulations in the liver of Atlantic salmon, particularly in biological pathways related to protein synthesis. The interplay between DNA methylation and gene expression in these pathways may play an important role in the mechanisms underlying growth performance affected by 1C metabolism.

Micronutrient supplementation affects DNA methylation in male gonads with potential intergenerational epigenetic inheritance involving the embryonic development through glutamate receptor-associated genes.

BACKGROUND: DNA methylation has an important role in intergenerational inheritance. An increasing number of studies have reported evidence of germline inheritance of DNA methylation induced by nutritional signals in mammals. Vitamins and minerals as micronutrients contribute to growth performance in vertebrates, including Atlantic salmon (Salmo salar), and also have a role in epigenetics as environmental factors that alter DNA methylation status. It is important to understand whether micronutrients in the paternal diet can influence the offspring through alterations of DNA methylation signatures in male germ cells. RESULTS: Here, we show the effect of micronutrient supplementation on DNA methylation profiles in the male gonad through a whole life cycle feeding trial of Atlantic salmon fed three graded levels of micronutrient components. Our results strongly indicate that micronutrient supplementation affects the DNA methylation status of genes associated with cell signalling, synaptic signalling, and embryonic development. In particular, it substantially affects DNA methylation status in the promoter region of a glutamate receptor gene, glutamate receptor ionotropic, NMDA 3A-like (grin3a-like), when the fish are fed both medium and high doses of micronutrients. Furthermore, two transcription factors, histone deacetylase 2 (hdac2) and a zinc finger protein, bind to the hyper-methylated site in the grin3a-like promoter. An estimated function of hdac2 together with a zinc finger indicates that grin3a-like has a potential role in intergenerational epigenetic inheritance and the regulation of embryonic development affected by paternal diet. CONCLUSIONS: The present study demonstrates alterations of gene expression patterns and DNA methylation signatures in the male gonad when Atlantic salmon are fed different levels of micronutrients. Alterations of gene expression patterns are of great interest because the gonads are supposed to have limited metabolic activities compared to other organs, whereas alterations of DNA methylation signatures are of great importance in the field of nutritional epigenetics because the signatures affected by nutrition could be transferred to the next generation. We provide extensive data resources for future work in the context of potential intergenerational inheritance through the male germline.

Metabolic and molecular signatures of improved growth in Atlantic salmon (Salmo salar) fed surplus levels of methionine, folic acid, vitamin B6 and B12 throughout smoltification.

A moderate surplus of the one carbon (1C) nutrients methionine, folic acid, vitamin B6 and B12 above dietary recommendations for Atlantic salmon has shown to improve growth and reduce hepatosomatic index in the on-growing saltwater period when fed throughout smoltification. Metabolic properties and molecular mechanisms determining the improved growth are unexplored. Here, we investigate metabolic and transcriptional signatures in skeletal muscle taken before and after smoltification to acquire deeper insight into pathways and possible nutrient­gene interactions. A control feed (Ctrl) or 1C nutrient surplus feed (1C+) were fed to Atlantic salmon 6 weeks prior to smoltification until 3 months after saltwater transfer. Both metabolic and gene expression signatures revealed significant 1C nutrient-dependent changes already at pre-smolt, but differences intensified when analysing post-smolt muscle. Transcriptional differences revealed lower expression of genes related to translation, growth and amino acid metabolisation in post-smolt muscle when fed additional 1C nutrients. The 1C+ group showed less free amino acid and putrescine levels, and higher methionine and glutathione amounts in muscle. For Ctrl muscle, the overall metabolic profile suggests a lower amino acid utilisation for protein synthesis, and increased methionine metabolisation in polyamine and redox homoeostasis, whereas transcription changes are indicative of compensatory growth regulation at local tissue level. These findings point to fine-tuned nutrient­gene interactions fundamental for improved growth capacity through better amino acid utilisation for protein accretion when salmon was fed additional 1C nutrients throughout smoltification. It also highlights potential nutritional programming strategies on improved post-smolt growth through 1C+ supplementation before and throughout smoltification.

Micronutrient supplementation affects transcriptional and epigenetic regulation of lipid metabolism in a dose-dependent manner.

Micronutrients (vitamins and minerals) have been less well studied compared to macronutrients (fats, proteins, and carbohydrates) although they play important roles in growth, metabolism, and maintenance of tissues. Hence, there is growing interest to understand the influence of micronutrients across various aspects in nutritional research. In the last two decades, aquaculture feeds have been shifted to containing more plant-based materials to meet the increasing demand and maintain the sustainability in the industry. A recent whole life cycle feeding trial of Atlantic salmon (Salmo salar) with graded levels of micronutrient packages has concluded that the levels of several B-vitamins and microminerals need to be increased from the current recommendation levels for optimal growth and fish welfare when plant-based diets are used. Here, we show the effect of micronutrient supplementation on hepatic transcriptional and epigenetic regulation in a dose dependent manner. . Specifically, our aim is to reveal the mechanisms of altered cell metabolism, which results in improved growth performance by micronutrient surpluses, at gene expression and DNA methylation levels. Our results strongly indicate that micronutrient supplementation suppresses gene expression in lipid metabolism in a dose-dependent manner and broadly affects DNA methylation in cell-adhesion and cell-signalling. In particular, it increases DNA methylation levels on the acetyl-CoA carboxylase alpha promoter in a concentration-dependent manner, which further suggests that acetyl-CoA carboxylase alpha is an upstream epigenetic regulator controlling its downstream lipid biosynthesis activities. This study demonstrates a comprehensive analysis to reveal an important role of micronutrients in lipid metabolism through epigenetic control of gene expression.

Parental Selenium Nutrition Affects the One-Carbon Metabolism and the Hepatic DNA Methylation Pattern of Rainbow Trout (Oncorhynchus mykiss) in the Progeny.

Selenium is an essential micronutrient and its metabolism is closely linked to the methionine cycle and transsulfuration pathway. The present study evaluated the effect of two different selenium supplements in the diet of rainbow trout (Onchorhynchus mykiss) broodstock on the one-carbon metabolism and the hepatic DNA methylation pattern in the progeny. Offspring of three parental groups of rainbow trout, fed either a control diet (NC, basal Se level: 0.3 mg/kg) or a diet supplemented with sodium selenite (SS, 0.8 mg Se/kg) or hydroxy-selenomethionine (SO, 0.7 mg Se/kg), were collected at swim-up fry stage. Our findings suggest that parental selenium nutrition impacted the methionine cycle with lower free methionine and S-adenosylmethionine (SAM) and higher methionine synthase (mtr) mRNA levels in both selenium-supplemented treatments. DNA methylation profiling by reduced representation bisulfite sequencing (RRBS) identified differentially methylated cytosines (DMCs) in offspring livers. These DMCs were related to 6535 differentially methylated genes in SS:NC, 6890 in SO:NC and 7428 in SO:SS, respectively. Genes with the highest methylation difference relate, among others, to the neuronal or signal transmitting and immune system which represent potential targets for future studies.

The effect of micronutrient supplementation on growth and hepatic metabolism in diploid and triploid Atlantic salmon (Salmo salar) parr fed a low marine ingredient diet.

The effects of low marine ingredient diets supplemented with graded levels (L1, L2, L3) of a micronutrient package (NP) on growth and metabolic responses were studied in diploid and triploid salmon parr. Diploids fed L2 showed significantly improved growth and reduced liver, hepatic steatosis, and viscerosomatic indices, while fish fed L3 showed suppressed growth rate 14 weeks post feeding. In contrast, dietary NP level had no effect on triploid performance. Whole body mineral composition, with exception of copper, did not differ between diet or ploidy. Whole fish total AAs and N-metabolites showed no variation by diet or ploidy. Free circulating AAs and white muscle N-metabolites were higher in triploids than diploids, while branch-chained amino acids were higher in diploids than triploids. Diploids had higher whole body α-tocopherol and hepatic vitamins K1 and K2 than triploids. Increased tissue B-vitamins for niacin and whole-body folate with dietary NP supplementation were observed in diploids but not triploids, while whole body riboflavin was higher in diploids than triploids. Hepatic transcriptome profiles showed that diploids fed diet L2 was more similar to that observed in triploids fed diet L3. In particular, sterol biosynthesis pathways were down-regulated, whereas cytochrome P450 metabolism was up-regulated. One­carbon metabolism was also affected by increasing levels of supplementation in both ploidies. Collectively, results suggested that, for optimised growth and liver function, micronutrient levels be supplemented above current National Research Council (2011) recommendations for Atlantic salmon when fed low marine ingredient diets. The study also suggested differences in nutritional requirements between ploidy.

Parental micronutrient deficiency distorts liver DNA methylation and expression of lipid genes associated with a fatty-liver-like phenotype in offspring.

Micronutrient status of parents can affect long term health of their progeny. Around 2 billion humans are affected by chronic micronutrient deficiency. In this study we use zebrafish as a model system to examine morphological, molecular and epigenetic changes in mature offspring of parents that experienced a one-carbon (1-C) micronutrient deficiency. Zebrafish were fed a diet sufficient, or marginally deficient in 1-C nutrients (folate, vitamin B12, vitamin B6, methionine, choline), and then mated. Offspring livers underwent histological examination, RNA sequencing and genome-wide DNA methylation analysis. Parental 1-C micronutrient deficiency resulted in increased lipid inclusion and we identified 686 differentially expressed genes in offspring liver, the majority of which were downregulated. Downregulated genes were enriched for functional categories related to sterol, steroid and lipid biosynthesis, as well as mitochondrial protein synthesis. Differential DNA methylation was found at 2869 CpG sites, enriched in promoter regions and permutation analyses confirmed the association with parental feed. Our data indicate that parental 1-C nutrient status can persist as locus specific DNA methylation marks in descendants and suggest an effect on lipid utilization and mitochondrial protein translation in F1 livers. This points toward parental micronutrients status as an important factor for offspring health and welfare.

Impact of dietary selenium on methylmercury toxicity in juvenile Atlantic cod: a transcriptional survey.

Selenium (Se) and its derivatives are known to have protective effects against mercury (Hg) toxicity in mammals. In this study we wanted to evaluate whether Se co-exposure affect the transcription of methylmercury (MeHg) toxicity-relevant genes in early life stages of fish. Juvenile Atlantic cod were exposed to regular feed (control), Se-spiked feed (3mg Se kg(-1)), MeHg-spiked feed (10mg Hg kg(-1)) or to Se- and MeHg-spiked feed (3mg Se kg(-1) and 10mg Hg kg(-1), respectively) for ten weeks. Liver tissue was harvested for transcriptional analysis when the fish were weighing 11.4 ± 3.2g. Accumulated levels of Hg in liver of the two groups of fish exposed to MeHg were 1.5mg Hg kg(-1) wet weight, or 44-fold higher than in the control group, while the Se concentrations differed with less than 2-fold between the fish groups. Selenium co-exposure had no effect on the accumulated levels of Hg in liver tissue; however, MeHg co-exposure reduced the accumulated level of Se. Dietary exposure to MeHg had no effect on fish growth. Interaction effects between Se and MeHg exposure were observed for the transcriptional levels of CAT, GPX1, GPX3, NFE2L2, UBA52, SEPP1 and DNMT1. Significant effects of MeHg exposure were seen for DNMT1 and PPARG, while effects of Se exposure were seen for GPX4B and SEPP1A, as well as for DNA methyltransferase activity. The transcriptional results suggest, by considering up-regulation as a proxy for negative impact and at the tested concentrations, a pro-oxidative effect of Se co-exposure with MeHg, rather than an antioxidative effect.

Ontogenetic expression of maternal and zygotic genes in Atlantic cod embryos under ambient and thermally stressed conditions.

The embryonic stages of Atlantic cod (Gadus morhua) are especially sensitive to incubation temperature. The purpose of the present study was to follow the ontogenetic expression of selected genes of maternal (pou2 and nanog) and zygotic origin (hsp70, hsp90α and stip1), in Atlantic cod embryos under ambient and thermally stressed conditions. The study also investigated how reference genes can be applied to studies on embryonic development, when maternal genes are degraded and the zygotic transcription stabilizes. Three batches of eggs were reared and gene expression profiles from the reference and target genes were determined. The embryos were reared at ambient 6 °C, and 10 °C for continuous long-term and acute short-term heat exposure. Both pou2 and nanog showed reduced expression whereas the zygotic and reference genes showed increased expression until stabilizing at gastrulation, when a normalized ontogenetic expression profile of target genes could be generated. pou2 and nanog were not affected by thermal stress. In contrast, hsp70 and hsp90α were upregulated after short-term heat exposure at the early blastula (hsp70 only), late blastula, 50% epiboly and 90% epiboly stages (hsp90α only). Long-term heat exposure of Atlantic cod embryos upregulated both hsp70 (90% epiboly) and hsp90α (90% epiboly and 20-somites). The results suggest that a cellular defense mechanism is activated even in the earliest stages of embryonic development, a period critical to developmental temperature.